Organ-on-a-chip (OoC) platforms have emerged as promising tools for modeling human tissues and organ systems, bridging the gap between traditional cell culture and in vivo pre-clinical models [1]. These OoC systems enable precise control over biochemical and mechanical cues and are valuable in facilitating drug discovery and unveiling disease mechanisms [[2], [3], [4]]. Despite their advantages, many OoC systems rely on synthetic or tumor-derived extracellular matrices (ECM) like Matrigel, which contains undefined tumorigenic components and lacks tissue-specific characteristics [5]. Specifically, more than 96 % of total matrisome proteins in Matrigel are glycoproteins, with laminin-111 among the top ten most abundant proteins. However, in mature tissues laminin-111 is not prominent, indicating a significant compositional mismatch between Matrigel and native ECM [6]. To address these limitations, tissue-specific decellularized ECM (dECM) has gained attention as a biomaterial that better preserves native biochemical composition, maintains three-dimensional (3D) architecture, and provides essential biological cues for cell adhesion, proliferation, and differentiation [7,8]. In the last decade, there has been a growing interest in using dECM to enhance tissue biomimicry for microtissue engineering [9]. For instance, Giobbe and colleagues demonstrated that human epithelial organoids, including small intestinal, liver duct, and hepatocyte organoids, cultured in intestine-derived dECM hydrogel exhibit a more stable transcriptomic profile compared to those in Matrigel [10]. In the same study, human pancreatic and intestinal organoids cultured in either dECM hydrogels or Matrigel showed comparable engraftment efficiency in vivo. Despite these advances in organoid technologies, the integration of dECM into OoC microfluidic devices for oral mucosa (OM) tissue mimicry remains underexplored. OM consists of a squamous stratified epithelium supported by an underlying connective tissue layer, the lamina propria [11,12]. The lamina propria mainly contains fibroblasts within a collagenous ECM rich in blood vessels, which contribute to nutrient supply, epithelial integrity and wound healing [13]. Therefore, to accurately replicate a full thickness OM, it is essential to develop a multi-layered oral mucosa microtissue with a dECM-based hydrogel that mimics the properties of lamina propria. This study addresses this gap by developing a hybrid hydrogel composed of OM dECM to fabricate a full-thickness multi-layered oral mucosa-on-a-chip (OMoC).

OMoC fluid flow devices have gained attention as platforms for investigating interactions between epithelial surfaces and oral pathogens [14,15]. Our research group recently developed a millifluidic-based OoC platform, which integrated bioprinted oral epithelia sheets with dynamic fluid flow to simulate chemotherapy-induced damage caused by 5-fluorouracil (5FU) [16]. This platform successfully recapitulated a multilayered stratification in the epithelium, and upon 5-FU exposure, cells secreted pro-inflammatory cytokines, a hallmark of oral mucositis. However, a key limitation of our OM OoC model was the absence of a lamina propria compartment containing fibroblasts and endothelial cells to trigger angiogenesis. To support and enhance the physiological relevance of this cellularized submucosa compartment, an appropriate ECM is essential. Previous studies of Muniraj et al., Gard et al., and Rahimi et al. used fibrin, basement membrane extract (BME), and collagen I as matrix components to mimic the lamina propria of the OM [[17], [18], [19]]. For instance, Muniraj et al. developed a full-thickness gingiva-on-a-chip using a fibrin-based matrix. However, fibrin matrix possesses several limitations including rapid degradation, shrinkage behavior, and pro-inflammatory effects, which limits its translational potential [20,21]. While these matrices provide structural support and facilitate cellular functions, they fail to recapitulate the complex biochemical composition of native OM [22,23]. As a result, these models may not accurately reflect the in vivo microenvironment, limiting their translational relevance. Therefore, there is a research need to develop a more physiologically relevant ECM substitute to improve the fidelity of the lamina propria in OMoC devices. Given the distinct composition and architecture of the OM, creating a dECM-based biomaterial tailored to this tissue is critical to advance towards OMoC microfluidic devices with higher complexity in terms of cellular diversity and collagenous matrix content.

Herein, this study aims to: (1) develop and characterize dECM derived from porcine OM, (2) fabricate a hybrid hydrogel by combining OM dECM with hyaluronic acid (HA) and alginate (Alg) to enhance injectability and bioactivity, and (3) develop a microfluidic OMoC model with epithelial and lamina propria layers. Moreover, the following null hypotheses were tested: (1) There is no difference in NOK, HGF, and HUVEC viability and proliferation between OM dECM hydrogels and Matrigel/BME; and (2) There is no difference in the developed oral mucosa tissue architecture in vitro between OM dECM–HA/Alg hybrid hydrogels, plain OM dECM hydrogels, and Matrigel.